Dual function of baicalin in nsPEFs-treated hepatocytes and hepatocellular carcinoma cells for different death pathway and mitochondrial response

Nanosecond pulsed electric fields (nsPEFs) is emerged as a potential curative modality to ablate hepatocellular carcinoma (HCC). The application of local ablation is usually limited by insufficiency of liver function. While baicalin, a flavonoid isolated from Scutellaria baicalensis Georgi, has been proven to possess both anti-tumor and protective effects.

Int J Med Sci 2019, Vol 16 1271

International Journal of Medical Sciences

2019; 16(9): 1271-1282 doi: 10.7150/ijms.34876

Research Paper

Dual-function of Baicalin in nsPEFs-treated Hepatocytes and Hepatocellular Carcinoma cells for Different Death Pathway and Mitochondrial Response

Yubo Wang1*, Shengyong Yin1*, Yuan Zhou1*, Wuhua Zhou1,2, Tianchi Chen1, Qinchuan Wu1, Lin Zhou1 ,

1 Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province 310003, China

2 Department of hepatobiliary and pancreatic surgery, Taihe Hospital, Hubei University of Medicine, Hubei, China

*Contributed equally

 Corresponding authors: Shusen Zheng, Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, No 79 Qingchun Road, Hangzhou 310003, China Tel: +86 571 87236466; Email: shusenzheng@zju.edu.cn Lin Zhou, Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, No 79 Qingchun Road, Hangzhou 310003, China Tel: +86 571 87236601; Email: zhoulin99@zju.edu.cn

© The author(s) This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) See http://ivyspring.com/terms for full terms and conditions

Received: 2019.03.13; Accepted: 2019.08.03; Published: 2019.09.07

Abstract

Nanosecond pulsed electric fields (nsPEFs) is emerged as a potential curative modality to ablate

hepatocellular carcinoma (HCC) The application of local ablation is usually limited by insufficiency

of liver function While baicalin, a flavonoid isolated from Scutellaria baicalensis Georgi, has been

proven to possess both anti-tumor and protective effects Our study aimed to estimate different

responses of hepatic cancer cells and hepatocytes to the combination of nsPEFs and baicalin Cell

viability, apoptosis and necrosis, mitochondrial transmembrane potential (MTP) and reactive oxygen

species (ROS) were examined by CCK-8, FCM, JC-1 and fluorescent probe, respectively After

treatment by nsPEFs, most hepatocytes died by apoptosis, nevertheless, nearly all cancer cells were

killed through necrosis Low concentration of baicalin synergically enhanced nsPEFs-induced

suppression and necrosis of HCC cells, nevertheless, the application of baicalin protected normal

hepatocytes from the injury caused by nsPEFs, owing to elevating mitochondrial transmembrane

potential and reducing ROS generation Our work provided an advantageous therapy for HCC

through the enhanced combination treatment of nsPEFs and baicalin, with which could improve the

tumor-ablation effect and alleviate the injury of hepatic tissues simultaneously

Key words: Dual Function, Hepatocellular Carcinoma, Nanosecond pulsed electric fields, Baicalin, Mitochondrial

transmembrane potential

Introduction

Liver cancer, of which about 75%-85% cases are

hepatocellular carcinoma (HCC), is quite prevalent

and lethal worldwide [1] Less than 30% of HCC

patients have an opportunity to undergo surgery due

to poor physical condition, major vascular invasion or

shortage of organ supply For most cases of HCC,

local treatments, comprising trans arterial

chemoembolization (TACE), radiofrequency ablation

(RFA) and percutaneous ethanol injection (PEI), are

widely adopted due to unavailable resection of tumor [2-4] However, these local strategies are frequently limited by multiple complications, for instance, thermal and chemical injuries To surmount these defects, a novel treatment nanosecond pulsed electric fields (nsPEFs), which employs nanosecond duration electrical pulses with utmost voltage and field strength, has been lately developed to ablate solid tumor by non-thermal way [3] Instantaneous huge Ivyspring

International Publisher

power of nsPEFs triggers death of tumor cells but is

merely harmful to intrahepatic ducts [5] NsPEFs can

induce cell death through several mechanisms,

mainly including the reversible electroporation of

plasma membrane (PM) and mitochondria damage [6,

7] These high intensity pulses expand the membrane

permeability and ultimately permit small molecules

to penetrate the plasma membrane such as calcium or

dyes, for instance, propidium (PI) and trypan blue

(TB)[8, 9] In addition, the latest evidence has shown

that the application of nsPEFs with much shorter

pulse duration has more impact on intracellular

organelle than plasma membrane [10], which leads to

the dissipation of mitochondria transmembrane

potential [7] Furthermore, nsPEFs can trigger calcium

overload [11], stress responses [12], apoptosis [11, 13,

14] and diverse signal kinase pathways activation in

cancer cells [15-17], and the ablation effect of nsPEFs

has been validated on various malignancies including

hepatocellular carcinoma [18], melanoma [19],

pancreatic cancer [20], squamous cell carcinoma [21]

etc

Although nsPEFs can effectively ablate hepatic

tumors, it is inevitable for nsPEFs to damage normal

hepatic tissues, which might cause liver insufficiency

In order to improve the therapeutic effect of nsPEFs,

baicalin, the major flavonoid and main active

ingredient purified from traditional Chinese medicine

Scutellaria baicalensis Georgi, whose chemical

constitution is known [22], is employed Baicalin has

been reported as an effective agent exhibiting

multiple pharmacological functions, for instance,

anti-tumor, anti-inflammatory and anti-oxidation

[23-25] These pharmacological functions are depend

on the arrest of cell cycle, induction of apoptosis,

reduction of reactive oxygen species (ROS) and

stabilization of mitochondrial transmembrane

potential (MTP) [26, 27] Baicalin or baicalein, 90% of

which would convert into baicalin in blood, has been

reported to be lethal to hepatic tumor by suppressing

tumor migration and invasion, inducing apoptosis

and inhibiting tumor growth [28, 29]

Since the anti-tumor function of nsPEFs has

been validated, we hypothesized that the application

of nsPEFs could effectively ablate HCC and the

normal hepatic tissue damage within the range of

effective electric field could be prevented by agents,

such as baicalin In this study, low concentration of

baicalin was used after the application of nsPEFs to

enhance the tumor-elimination capability and protect

normal hepatocytes from the injury caused by nsPEFs

simultaneously The results demonstrated a dual

function that the combined therapy could inhibit HCC

cells more effectively by enhancing necrotic cell death

but alleviate the damage of normal hepatocytes by

preserving mitochondrial transmembrane potential and cleaning up cellular reactive oxygen species These findings elicited a potential clinical strategy to eliminate hepatocellular carcinoma more sufficiently while alleviating the damage of normal hepatic tissues and provided a conceivable clinical guidance for nsPEFs

Materials and Methods

Cell culture

Human normal hepatocyte line QSG-7701 and human hepatocellular carcinoma cell line MHCC-97H were purchased from the Chinese Academy of Science High metastatic HCC cell line HCC-LM3 was purchased from the Liver Cancer Institute, Zhongshan Hospital, Fudan University QSG-7701 cells were maintained in RPMI-1640 (Gibco-Invitrogen, Carlsbad, CA, USA) and MHCC-97H, HCC-LM3 cells were maintained in DMEM (Gibco-Invitrogen, Carlsbad, CA, USA), and both mediums were supplemented with 10% fetal bovine serum (FBS, SAFC Biosciences, Lenexa, KS, USA), 100 unit/ml penicillin and 100 mg/ml streptomycin (SigmaAldrich, St Louis, MO, USA)

Isolation and culture of primary mouse hepatocytes

The primary mouse hepatocytes were isolated from 28-day-old male C57BL/6 mice The mouse was first anaesthetized and the liver was perfused with Krebs-Ringer buffer and collagenase IV (Sigma Aldrich, St Louis, MO, USA) without calcium and magnesium Fibroblasts and liver non-parenchymal cells were removed through DMEM elution The primary mouse hepatocytes were seeded onto a collagen-coated plate and cultured with the special complete medium of primary mouse hepatocytes (Procell, Wuhan, China) (Figure 1D) All animal experiments were performed in accordance with protocols and regulations of the Experimental Animal Ethics Committee of the First Affiliated Hospital of Zhejiang University (Hangzhou, Zhejiang, China)

NsPEFs generator and nsPEFs application

NsPEFs generator’s essential parameter adjustment was shown in our previous study [35] (Figure 1A) Waveforms were monitored with a digital phosphor oscilloscope (DPO4054, Tektronix, USA, Figure 1C) equipped with a high voltage probe (P6015A, Tektronix, USA) Cells were harvested with trypsin (Gibco-Invitrogen, Carlsbad, CA, USA) and re-suspended in advance preparing medium to a concentration of 2.0×106 cells/ml Antibiotic free pulse mediums included RPMI-1640 containing 10% FBS for QSG-7701 and DMEM containing 10%FBS for

Int J Med Sci 2019, Vol 16 1273 MHCC-97H and HCC-LM3 1 ml of cell suspension

was placed into a 0.4 cm gap cuvette (Biosmith,

aluminum plate electrodes, Figure 1B) and exposed to

100ns, 1 HZ, 30, 40, 50, 60, 80 pulses at 15, 25 and 40

kV/cm electric field strength, respectively These

nsPEFs-treated cells (8000 cells/well) were seeded

into 96-well plates and incubated for 24h, then their

death/viability was detected by CCK-8 (Dojindo,

Kumamoto, Japan) assay

Baicalin exposure and combination treatment

of baicalin and nsPEFs

For baicalin treatment, cells were seeded into

96-well plates and treated by baicalin (Solorbio,

Beijing, China) with concentration of 0.1, 1, 10, 20, 40,

80, 160, 320 and 640 μM, respectively, for 24h or 48h,

and then their viability was measured with CCK-8

assay For combined treatment of baicalin and nsPEFs,

cells were first exposed to 40P, 15, 25 and 40 kv/cm

nsPEFs, then placed into 96-well plates (8000/well)

Cells were cultured for 6h to adhere to the plate and

then incubated with 0.625μM baicalin for 24h or 48h,

following by viability measurement with CCK-8

assay

Cell apoptosis and necrosis analysis

Cell apoptosis and necrosis was quantitatively measured using Annexin V-FITC apoptosis detection kit (Dojindo, Kumamoto, Japan) by flow cytometry (FCM) Cells were harvested and washed by PBS, then dyed by FITC and PI (8μl/ml) for 30 min in room temperature before detected by FCM Double-negative FITC-/PI-, single positive PI+, single positive FITC+ and double-positive FITC+/PI+ represented the living cells, mechanical injury cells, early phase apoptotic cells and late phase apoptotic or

necrotic cells, respectively

Western-blot assay

RIPA buffer was utilized in lysing cells All protein concentration was quantified by BCA method 25μg of proteins from each group were loaded on ExpressPLUSTMPAGE gels (GenScript, USA) and then transferred on PVDF membranes before incubated with primary antibodies (1:2000) overnight After incubation with HRP-conjugated secondary antibody (1:5000) for 2h, proteins were detected by EZ-ECL (Biological Industries, Israel) Anti-PAR (ab14459), anti-cleaved PARP-1 (ab32561) and anti-β-actin (ab8226) were purchased from Abcam

(Cambridge, UK)

Figure 1: Demonstration of nsPEFs generator (A), cuvettes (B), basic wave of nsPEFs (C) and primary mouse hepatocytes (D)

Mitochondrial transmembrane potential

measurement

The tetraethylbenzimidazolylcarbocyanine

iodide (JC-1) is a cationic dye that accumulates in

energized mitochondria Cells were harvested and

washed after handled by different treatment for 24h

For positive control, untreated cells were first mixed

with 3μl/ml CCCP and PBS for 1h in 37℃ before

mixed with 1μl/ml JC-1 (MultiScience, Hangzhou,

China) for 30min in 37℃ For other groups, 1μl/ml

JC-1 were mixed with cells and PBS for 30min in 37℃

Eventually, red fluorescence indicated by PE is

detected when JC-1 accumulation in mitochondria is

sustained by the normal cellular electrochemical

potential gradient, or green fluorescence indicated by

FITC is present when JC-1 is dispersed into cytoplasm

for the dissipation of MTP through FCM

Intracellular ROS detection by cell ROS

reagent

NsPEFs-treated cells were incubated in the

presence or absence of 0.625μM baicalin for 24h, and

then washed twice with PBS before stained with

2μl/ml CellROX Green Reagent (ThermoFisher, MA,

U.S.A) for 30 min in 37℃ and light-resistant

incubator Ultimately, the concentration of ROS was

measured by FCM and fluorescent microscopy,

during which the detectable bright green fluorescent

signal (at the light wave length of 488nm, or Alexa

Fluor 488-A) presented the amount of ROS

Cell Viability

Cells were placed into 96-well plates and

incubated with 10 μl CCK-8 solution at 37°C Each

sample was replicated 6 times After 1 hour, the

optical density was obtained at 450nm by a

spectrophotometer (ELx800; BioTek Instruments, Inc.,

Vermont, VT, U.S.A) The relative survival rate was

calculated by the ratio of OD values of experiment

group to OD values of control group

Statistical analysis

Raw data were normalized by Microsoft Excel

2010 and figures were generated by GraphPad Prism

5.0 (GraphPad Software, San Diego, CA, U.S.A)

Statistical analysis was performed with SPSS 16.0 for

windows (SPSS, Chicago, IL, U.S.A) Quantitative

variables were expressed as means ± SD For FCM

data, FlowJo V10 (FlowJo LLC, Ashland, OR, U.S.A)

was participated Student’s t-test, one-way ANOVA

and χ2 analysis were performed to analyze variance

Results were considered statistically significant at P <

0.05 All experiments were repeated three times

Results

Baicalin was more toxic to cancer cells while

less toxic to hepatocytes

Previous researches have proved that baicalin could suppress HCC cells including HepG2 and SMMC-7721 and has fewer side effects to normal hepatocytes, the Chang liver cell line [26, 28, 29] In our study, we first assessed the toxicity of baicalin on HCC cell lines MHCC-97H and HCC-LM3, normal human hepatic cell line QSG-7701 and primary mouse hepatocytes The survival rates of all cells are shown

in Figure 2A Although the inhibition was not significantly different at 24h or 48h after baicalin treatment in HCC cell lines, primary mouse hepatocytes were more sensitive to baicalin at 48h after treatment (P<0.05) In order to reduce the toxicity of baicalin on normal hepatocytes, 24h after baicalin treatment was chosen in the following mechanism researches This conclusion will be further corroborated in Figure 3 IC50 values of HCC cells was much higher than normal hepatocytes after 24h while

no statistical differences in IC50 values between HCC cells and normal hepatocytes after 48h (Figure 2C) Observations of differences between HCC cells and normal hepatocytes in IC50 values demonstrated that baicalin was more toxic to cancer cells than normal hepatocytes at 24h after baicalin treatment

Determination of appropriate nsPEFs parameters

To determine the appropriate parameters of nsPEFs for the combined treatment, a train of nsPEFs with the doses of 30, 40, 50, 60, 80 in pulse number and the strength of 15kv/cm, 25kv/cm, 40kv/cm in electric fields were applied The reason why we selected 30-80 in pulse number was the generator was set to generate 10 pulses as a group in order to stabilize the wave form and reduce pulse stretching

As shown in Figure 2B, the number of pulses was chosen at 40 pulses because the cell viability after nsPEFs treatment with 40kv/cm at more than 40 pulses in HCC-LM3 cell line was too low (about 10%) and that with 25kv/cm and 40kv/cm at 30 pulses in QSG-7701 cell line had no statistical difference Interestingly, although nsPEFs non-selectively killed cells, the tolerance of different cells to nsPEFs was in variety HCC cell line MHCC-97H seemed to be more susceptible to nsPEFs, while other cells exhibited no apparent difference, which indicated different cell structure might correlate with the different susceptibility to nsPEFs

Int J Med Sci 2019, Vol 16 1275

Figure 2: Effect of Baicalin and nsPEFs on cell viability of HCC cell lines and normal liver cells Different cells were treated with baicalin of indicated concentration

(A, C) for 24h or 48h and nsPEFs with indicated parameters (B) for 24h Viabilities were assessed by CCK-8 IC50, half maximal inhibitory concentration *P<0.05

Combined treatment of nsPEFs and baicalin

synergistically inhibited HCC cells whereas

protected normal hepatocytes

After the treatment of 40P, 25kv/cm nsPEFs

combined with a train of baicalin with the

concentration of 0-80μM on all cells within 24h or 48h,

it appeared that the concentration of 0.625μM was

more appropriate for baicalin to alleviate the injury of

nsPEFs on normal hepatocytes and enhance the

suppression of nsPEFs on HCC cells simultaneously

(Figure S1) To further explore the synergistic function

of combined application of nsPEFs and baicalin, all

cells were treated with 0.625μM baicalin and nsPEFs

with 40 pulses and 15, 25, 40 kv/cm electric field

strength for 24h and 48h In this regarding

experiment, effect difference (ED) value is assessed

according to the relative cell viability of the group

with nsPEFs treatment alone to that of the group with

combined treatment, and the positive or negative

value represented synergistic lethal or protective

effects, respectively Figure 3 shows that treatment

with 0.625μM baicalin alone had no impact on cell

viability within both cancer cells and hepatocytes

However, combined treatment of this relative low

concentration of baicalin and nsPEFs with 40 pulses,

25kv/cm caused striking inhibition on both MHCC-97H and HCC-LM3 cell lines compared with the treatment of nsPEFs alone, with the ED value of 20.75, 18.51 at 24h, respectively and 13.73, 15.45 at 48h, respectively (Figure 3A-B) Of interest, combined treatment of 0.625μM baicalin and nsPEFs with 40 pulses, a train of 15-40 kv/cm field strength for 24h and 48h lead to negative ED values within QSG-7701 cell line and primary mouse hepatocytes (Figure 3C-D) The absolute value of ED on cell line MHCC-97H, HCC-LM3, QSG-7701 and primary mouse hepatocytes at 24h were higher than that at 48h, which indicated that the synergism of combined treatment at 24h was more powerful than 48h and further confirmed the conclusion above that 24h was the more appropriate time point These observations suggested completely different lethal mechanisms caused by nsPEFs between HCC cells and normal

hepatocytes

NsPEFs was able to trigger distinct cell death modes

Since the ED value of 15kv/cm and 40kv/cm on all cells were too small and had no statistical difference, the appropriate electric field of nsPEFs was 25kv/cm In summary, the right parameter of

combined treatment in the following mechanism

experiments was 40 pulses, 25kv/cm for nsPEFs and

0.625μM for baicalin within 24h To explore the

underlying mechanisms of lethal and protective

effects, FCM was applied to evaluate the effect of

treatment of nsPEFs and/or baicalin on cell death As

shown in Figure 4A-H, the cell death (total number of

necrosis and apoptosis) triggered by nsPEFs was

enhanced by low concentration of baicalin within

MHCC-97H and HCC-LM3 cell lines whilst

suppressed within QSG-7701 cell line and primary

mouse hepatocytes In addition, the contribution of

combined treatment to ratio of necrotic cells to

apoptotic cells was facilitated within MHCC-97H and

HCC-LM3 cell lines whereas nearly stable within

QSG-7701 cell line and primary mouse hepatocytes

(Figure 4I-J), reflecting that there were two different

death modes between HCC cells and normal

hepatocytes

In order to confirm the potential distinct cell

death modes within HCC and normal liver cells, the

protein levels of Poly (ADP-ribose) (PAR) and cleaved

PAR polymerase-1 (cleaved PARP-1), which indicated

necrosis and apoptosis, respectively, were examined

by western-blot assay PAR is polymerized by

PARP-1, a downstream target of activated caspase 3,

within the nucleus [30, 31, 32] In accordance, PAR

amount was significantly increased within MHCC-97H and HCC-LM3 cell lines while hardly detected within QSG-7701 cell line and primary mouse hepatocytes after treatment of nsPEFs or combined treatment of nsPEFs and baicalin (Figure 4K) Moreover, the protein expression of cleaved PARP-1 was hardly examined within MHCC-97H and HCC-LM3 cell lines while remarkably elevated within QSG-7701 cell line and primary mouse hepatocytes after treatment of nsPEFs or combined treatment of nsPEFs and baicalin Besides, the protein expression

of PAR within HCC cells and cleaved PARP-1 within normal hepatocytes were higher and lower, respectively, after combined treatment than nsPEFs treatment alone Together, above observations mirrored that baicalin treatment was capable of increasing the number of necrotic cells within MHCC-97H and HCC-LM3 cell lines whereas reducing the number of apoptotic cells within QSG-7701 cell line and primary mouse hepatocytes, under the context of nsPEFs treatment These findings further suggested that, after the treatment of nsPEFs, HCC cell lines and normal hepatocytes exploited two distinct cell death modes, necrosis and apoptosis, respectively, which could be promoted and inhibited

by baicalin, respectively

Figure 3: Synergic effect of treatment of nsPEFs and/or baicalin with low concentration on cell viability Treatment of baicalin with concentration of 0.625μM was little harmful to HCC and normal liver cells HCC cell lines MHCC-97H (A), HCC-LM3 (B) and normal hepatocyte QSG-7701 (C), primary mouse hepatocytes (D) were treated

by baicalin with low concentration 0.625μM and/ or nsPEFs with parameter of 40P, 15, 25, 40kv/cm for 24h or 48h, followed by assessment of cell viability ED value, effect difference value The ED value were exhibited as mean value of triplicate independent experiments P, nsPEFs treatment; PBC, combined treatment of nsPEFs and baicalin

*P<0.05

Int J Med Sci 2019, Vol 16 1277

Figure 4: Baicalin enhanced cell necrosis but inhibited apoptosis under nsPEFs treatment Cell death of MHCC-97H, HCC-LM3, QSG-7701 cell lines and primary

mouse hepatocytes after treatment of baicalin and/ or nsPEFs for 24h was evaluated by flow cytometry (A-D) The percentage of cell death (E-H) or ratio of necrosis (or late apoptosis) to early apoptosis for the group with nsPEFs treatment alone (J) and the group with combined treatment (I) were quantified according to flow cytometry results The amount of cell death markers, PAR and cleaved PARP-1, were examined by western-blot assay (K) PMH: primary mouse hepatocytes; NC, cells without any treatment of nsPEFs

or baicalin as negative control; P, nsPEFs treatment; BC, baicalin treatment; PBC, combined treatment of nsPEFs and baicalin *P<0.05, **P<0.01

Baicalin suppressed nsPEFs-induced

mitochondrial transmembrane potential

dissipation within normal hepatocytes rather

than HCC cell lines

It has been evident that nsPEFs is able to

dissipate MTP and finally result in cell apoptosis [7]

while baicalin is protective to injured cells [27]

Therefore, it was reasonable for baicalin to inhibit

nsPEFs-mediated cell apoptosis in normal

hepatocytes QSG-7701 and primary mouse

hepatocytes through reducing MTP dissipation, but

not in HCC cell lines MHCC-97H and HCC-LM3 To

testify this hypothesis, the alteration of MTP was

examined by cationic lipophilic dye JC-1 within HCC

cells and normal hepatocytes after nsPEFs treatment

or combined treatment of nsPEFs and baicalin As

depicted in Figure 5, the transition of red to green

fluorescent signal instantaneously (at 0h) increased in

large extent, but decreased in 24h within both HCC

cells and normal hepatocytes after nsPEFs treatment

Furthermore, application of baicalin could

significantly enhance the process of decrease in

transition of red to green fluorescent signal within

primary mouse hepatocytes and cell line QSG-7701

rather than HCC cell lines MHCC-97H and HCC-LM3

at 24h These results demonstrated that the treatment

of low concentration of baicalin was able to suppress MTP dissipation triggered by nsPEFs application within normal hepatocytes QSG-7701 and primary mouse hepatocytes but not HCC cell lines MHCC-97H

and HCC-LM3

Baicalin cleared up generation of reactive oxygen species (ROS) after nsPEFs treatment

Since nsPEFs treatment was capable of triggering accumulation of ROS which could be cleared up by baicalin [33, 34], combined treatment of baicalin probably enabled normal hepatocytes to overcome the oxidative stress caused by nsPEFs and finally to escape from apoptosis To exactly detect the change of ROS amount after the treatment of nsPEFs or combination of nsPEFs and baicalin, a novel fluorogenic probe for ROS detection which could bind

to intracellular DNA as well was utilized As expected, nsPEFs treatment could significantly promoted ROS production, indicated by dramatic enhancement of green fluorescent signal, which showed the decreasing trend after combined application of nsPEFs and baicalin, especially within

normal hepatocytes QSG-7701 and primary mouse

hepatocytes (Figure 6A-D) These observations were

further confirmed by FCM results (Figure 6E-L),

reflecting that baicalin was able to clear up

intracellular ROS accumulation to some extent, in

particular within normal hepatocytes

Discussion

NsPEFs is capable of ablating malignancies

through various mechanisms, including inducing

apoptosis, increasing PM permeability, activating

several kinase pathways, etc.[11-17, 19, 35-38] NsPEFs

could efficiently ablate tumor lesions through the

low-thermal effect [39], but still along with challenges

including tumor recurrence or incidences of injury or

inflammation within normal tissues [18, 40] The

current study demonstrated that nsPEFs was able to

dramatically kill HCC cells and normal hepatocytes at

the same parameters In addition, a novel strategy of

combined treatment with nsPEFs and low concentration of baicalin displayed an inspiring therapeutic outcome that facilitated the suppression

of HCC cells whereas attenuated injury of normal hepatocytes through reducing the nsPEFs-triggered MTP dissipation and ROS accumulation

NsPEFs treatment could cause several cell death modes, consisting of apoptosis, autophagy related apoptosis or necrosis, which were largely dependent

on cell types, culture status or nsPEFs parameters [41, 42] Despite that, few evidences clarify the difference

of cell death modes implemented by different cell lines under the treatment of nsPEFs with the same parameter [43] Here, two distinct cell death modes were uncovered within HCC cell lines and normal hepatocytes Both results of FCM and protein amount

of death markers by immunoblotting showed that cell death of HCC cell lines and normal hepatocytes mainly relied on necrosis and apoptosis, respectively

Figure 5: Different functions of baicalin on nsPEFs-mediated dissipation of mitochondria transmembrane potential between normal hepatocytes and HCC cell lines Both HCC cell lines MHCC-97H, HCC-LM3 and normal hepatocytes QSG-7701, primary mouse hepatocytes were treated by baicalin with concentration

0.625μM and/ or nsPEFs with parameter of 40P, 25kv/cm for indicated time, and then their mitochondria transmembrane potential was detected using JC-1 assay through FCM

PE and FITC represented red and green fluorescent signal detected by FCM, respectively (A-D) The quantification of transition of red fluorescent signal to green fluorescent signal (E-H) NC, cells without any treatment of nsPEFs or baicalin as negative control; P, nsPEFs treatment; BC, baicalin treatment; PBC, combined treatment of nsPEFs and baicalin *P<0.05

Int J Med Sci 2019, Vol 16 1279 Specifically, the characteristics of dead HCC cells

were double positive PI(+)FITC(+) by FCM and

increase in protein amount of PAR by immunoblot,

differing from single positive FITC(+) and increased

protein level of cleaved PARP-1 by immunoblot

within normal hepatocytes Similarly, dependence on

necrosis of malignant cells after nsPEFs treatment was

unveiled in the study of nsPEFs treatment on

lymphadenoma cell line U937 [41] However, another

inconsistent evidence revealed that melanoma cancer

cells are destructed by nsPEFs through apoptosis [19]

This discrepancy might be caused by the different

parameters of diverse nsPEFs generator, for instance,

pulse width, electric fields strength, pulse stretching

or pulse frequency etc Furthermore, it remained

further confirmation that these distinct death modes

also existed in malignant and normal cells originated

from other organs

The dual-function of baicalin on anti-tumor and

anti-oxidation [23, 29] clued that baicalin might be an

ideal pharmacological agent for enhancing ablation

effect of tumor and alleviating complications by nsPEFs treatment Of interest, combined treatment of low concentration of baicalin exhibited the ability to promote the death of HCC cells and reduce the death

of normal hepatocytes caused by nsPEFs treatment Sole application of baicalin with low concentration was quite safe for cell survival (Figure 2-3), differing from killing effects of baicalin with a higher concentration on tumor cells reliable on apoptosis pathway [28, 29] However, treatment of baicalin with higher concentration would be harmful to the survival

of normal hepatocytes, which could possibly be accountable for the priority of cell death pathways over anti-oxidation or cell survival protection pathways Due to distinct cell death modes induced

by nsPEFs, necrosis within HCC cells and apoptosis within normal hepatocytes, difference in loss of plasma membrane integrity would probably allow different intracellular accumulation of agents [41], such as baicalin, and eventually affect the fate of different cells

Figure 6: Baicalin cleared up nsPEFs-induced ROS accumulation within normal hepatocyte HCC cell lines MHCC-97H, HCC-LM3 and normal hepatocyte

QSG-7701, primary mouse hepatocytes were treated by baicalin with concentration 0.625μM and/ or nsPEFs with parameter of 40P, 25kv/cm for indicated time, and then their ROS production was detected by CellROX Green Reagent through fluorescent microscopy (A-D) or FCM (E-H) Transition of red fluorescent signal to green fluorescent signal was the quantified (I-L) NC, cells without any treatment of nsPEFs or baicalin as negative control; P, nsPEFs treatment; BC, baicalin treatment; PBC, combined treatment of nsPEFs and baicalin *P<0.05 **P<0.01

Figure 7: Schematic illustration of mechanism that nsPEFs-triggered cell death was suppressed within normal hepatocytes whilst facilitated within HCC cells by baicalin

Intracellular ROS accumulation and MTP

dissipation, two typical features of cells after

treatment of nsPEFs, play key role in cell death [35]

This was substantiated by our findings including

spontaneous ROS production with a large amount

and MTP dissipation with different degrees within

both HCC and normal liver cells Given roles in

anti-oxidant stress [22, 23] and the close correlation

with ROS, MTP and apoptosis [44-46], baicalin

possesses the potential to clear up nsPEFs-induced

intracellular ROS accumulation and stabilize MTP

and ultimately overcomes cell death of normal

hepatocytes under nsPEFs treatment As expected,

our results demonstrated that combined usage of

baicalin with low concentration suppressed the

dissipation of MTP and reduced the accumulation of

ROS within normal hepatocytes QSG-7701 and

primary mouse hepatocytes after the application of

nsPEFs, in agreement with the effect of baicalin on the

reverse of ultraviolet radiation-induced oxidative

damage [27] However, treatment of baicalin with low

concentration could induce a descending trend of

nsPEFs-induced ROS generation but not MTP

recovery within HCC cell line HCC-LM3, supporting

that baicalin could clear up ROS non-selectively [47]

Additionally, MTP dissipation within necrotic cells

[48], resulting in a decrease of ATP and dysfunction of

mitochondria, was tougher to overcome [49] The

present observations confirmed that nsPEFs-induced

MTP dissipation within necrotic HCC cells was more

difficult to recover despite the reduction of ROS

accumulation to certain extend within HCC cells by low concentration of baicalin This could explain that baicalin could not attenuate nsPEFs-induced cell death of HCC cell lines

In conclusion, HCC cells and normal hepatocytes are killed by nsPEFs mainly through necrosis and apoptosis, respectively NsPEFs-induced cell deaths

of HCC and normal liver cells can be promoted and attenuated by baicalin with low concentration, respectively, and baicalin prevents normal hepatocytes from damage by nsPEFs largely through clearing up ROS production and stabilizing MTP (Figure 7) Our findings provide an advantageous therapy for HCC that combined treatment of nsPEFs and baicalin could improve the tumor-ablation effect

as well as reduce complications of the clinical application of nsPEFs

Abbreviations

HCC: hepatocellular carcinoma; nsPEFs: nanosecond pulsed electric fields; ROS: reactive oxygen species; MTP: mitochondrial transmembrane potential; ATP: adenosine triphosphate; PM: plasma membrane; TACE: trans arterial chemoembolization; RFA: radiofrequency ablation; PEI: percutaneous ethanol injection; CCK-8: cell counting kit 8; FCM: flow cytometry; PAR: poly (ADP-ribose)

Supplementary Material

Supplementary figure

http://www.medsci.org/v16p1271s1.pdf